The broad objectives of this proposal are to evaluate the role of heat-induced changes in specific intracellular ion concentrations on cell killing and thermotolerance. Specifically, flow cytometry and image analysis methods will be used to measure changes in intracellular pH, and intracellular free calcium, sodium and potassium ions in normal Chinese hamster ovary (CHO) cells and two mutant CHO cell lines. The mutants include an acid-tolerant cell line (PHV2) and a cell line that can be stained with indo-1 for calcium measurements. The cells will be heated for various treatments at 42 degrees C and 45 degrees C in both normal and low pH medium. The ion concentrations will be measured with fluorescent dyes using ratioing techniques to eliminate variations due to cell volume and dye concentrations. Intracellular pH will be measured with SNARF-1, intracellular free calcium will be measured with indo-1, and sodium and potassium ions will be measured with the new dyes SBFI and PBFI. Amiloride, ouabain, and sodium-free medium will be used to block ion pumps to prevent specific ion regulation in some experiments. An important feature of this proposal is that the cells will be heated directly on the cell sorter and the fluorescence microscope stage. This makes it possible to evaluate changes in these ions during heating. Another critical feature is that cells will be sorted based upon changes in intracellular pH or free calcium and plated for survival. Thus, instead of relying on correlations, a direct test of the cause-effect relationship between ion concentrations and survival can be made. The combination of flow cytometry and image analysis methods give complementary information that will provide a more detailed understanding of the role of these ions in cellular responses to hyperthermia. The importance of these studies for the clinical use of hyperthermia is that many tumors are known to have a low pH and low pH sensitizes cells to hyperthermia. However, the specific mechanisms are unknown. Also, the development of thermotolerance may limit the usefulness of hyperthermia as a therapy. Thus, a better understanding of the mechanisms of cell killing and thermotolerance may lead ultimately to better cancer therapy.